A proven means for the prophylaxis of viral diseases is immunization, the vaccination of the body against the viruses. In earlier times viral diseases which occurred in epidemic fashion such as e.g. smallpox and polio were frequently able to be checked by means of vaccination. In order to achieve such an active immunization against a virus the body, more precisely the immune system, is exposed to a viral antigen. This can take place e.g. by injecting inactivated or attenuated viruses or also parts, e.g. of proteins of the virus. The human immune system is basically composed of two different partial systems, the cellular immunological response (T cells) and the humoral immunological response (B cells, antibodies). In the case of a reinfection with a virus the humoral immunological response, imparted by B cells, represents the most important defense mechanism.
The immune system of the body reacts to the antigen with the formation of specific antibodies which recognize and bind the antigen and therewith the virus, thus initiating its inactivation. In addition, so-called "memory cells" are formed, that is, special lymphocytes which are activated upon a later infection with "their" virus or "their antigen" and stimulate the immune system very rapidly to synthesize large amounts of the antigen-specific antibodies. In this manner the immune system can react significantly more rapidly to a viral attack than if it had never been confronted previously by the corresponding antigen.
However, the use of attenuated viruses for vaccination can be problematic in as far as it can not be excluded that the viruses used become virulent in the body again and an outbreak of the viral disease occurs. It is preferable for this reason to use an isolated protein of the virus as immunogen. For this, it is best to use a surface protein of the virus since it is normally readily accessible to the humoral immunological response. Ideally, only a part of a surface protein is used, namely, that part which is located externally on the virus surface, the so-called ectodomain, since in the case of the intact virus only this part is accessible to antibodies. However, care should be taken in the production of a protein domain to be used for vaccination against a virus that it should have the native form to the extent possible since otherwise there is the danger that antibodies are formed which do not recognize the native protein and therewith the virus and are therewith ineffective for neutralization. Such molecules or molecule parts which are recognized on the basis of their three-dimensional structure by antibodies are called conformational or structural epitopes and those which are recognized by their amino-acid sequence are called sequential epitopes. It turns out more and more that there are only very few sequential epitopes and that most antigenic epitopes are structural, that is, they must exhibit the correct three-dimensional folding of the polypeptide chain in order to be recognized by corresponding antibodies. "Native" means in this context that the spacial structure of the protein domain used is identical or nearly identical to the structure of the corresponding protein range occurring naturally on or in the virus. This includes, if necessary, the presence of oligomery and/or glycosylation of the protein domains. A significant criterion for this is the fact that antibodies which were formed against an isolated native, viral protein domain also recognize and bind the virus and the corresponding native protein in the virus.
The human immunodeficiency virus HIV, a retrovirus, poses great problems today. Since it was identified in 1984 as the cause of AIDS great efforts have been undertaken to produce a vaccine against this virus too. One has recently succeeded in successfully immunizing monkeys with attenuated SIV viruses (simian immunodeficiency virus), close relatives of HIV, which allows the supposition that humans could also be successfully immunized against HIV by means of attenuated HIV. The problem entailed by attenuated viruses as vaccines has already been addressed. In the case of a deadly disease like AIDS this risk that the attenuated virus becomes virulent again can not be accepted, even if it is very small. See in this regard also: Koff, W. C. and Hoth, D. F. in the journal "Science", 241, 426-432 (1988).
For this reason a great part of the research about HIV vaccines is concentrated on using individual proteins, especially the surface protein of the virus, the so-called gp 160, also called env-glycoprotein, or fragments thereof as antigen. (A general survey is offered by the article "AIDS-Impfstoffe" [German--AIDS Vaccines] by T. H. Matthews et al. in the journal "Spektrum der Wissenschaft", 12, 134-142 (1988).)
The env-glycoprotein of HIV, whose amino-acid sequence is known (Ratner et al., "Nature" , 313, 277-284 (1985)), is first synthesized in the host cell as a highly glycosylated precursor protein gp 160 and subsequently processed in a Golgi's body to the two domains gp120 and gp41. The gp120 domain, which is located externally on the virus membrane, is responsible for the binding to the cellular receptor CD4 and both gp120 and precursor protein gp160 bind with high affinity to this CD4 receptor. Gp41, which exhibits a transmembrane domain, participates in the fusion of viral and cellular membrane during the penetration of the virus into the cell, probably by means of the interaction of a "fusion peptide" located on the amino terminus of gp 41, with the lipid double layer of the host cell membrane.
The approach of using only the external part of the env glycoprotein for the development of an HIV vaccine appears to be quite promising. This is based on the consideration already mentioned that the part of the glycoprotein which is exposed to the outside is the most easily accessible to the humoral immunological response and therefore an immunization against this ectodomain is the most promising.
The obtention of the ectodomain of the HIV surface protein can take place in various ways. It can e.g. be "cut off" by mechanical, chemical or enzymatic methods from the virus coating. The danger is great thereby that the protein is denatured and results in the formation of antibodies which recognize the native surface protein and therewith the virus only poorly or not at all.
Robey et al. ("Proc. Natl. Acad. Sci. USA", 83, 7023-7027 (1986)) obtain gp120 e.g. by removing the protein from the surface of HIV-infected cells by means of the rather strong detergent Triton X100. Even if the authors call their preparation "native gp120", it is doubtful in light of the detergent used and the purification methods used whether the gp120 obtained in this manner is actually present in native and oligomeric form.
A genetic engineering method consists in that a stop codon is inserted quasi as "theoretical breakage site" shortly before the start of the gp41 gene. During the expression of the gene in suitable cells the position of the stop codon then determines the position of the carboxy terminal end of the ectodomain of the surface protein, that is, only the external part of the surface protein, the domain gp 120, is synthesized. However, when synthesized in this manner gp 120 is present as a monomer, which does not correspond to the natural conditions on the virus since the env glycoprotein is present, like most viral glycoproteins in native form, as oligomer. Frequently, these proteins are trimers; it was recently able to be demonstrated for the HIV-1-env glycoprotein that it is a tetramer in native form (Schawaller, M. et al., "Virology", 172, 367-369 (1989). This explains why monomeric gp120 does not result in suitable antibodies and can therewith be poorly utilized as vaccine. Even experiments of the inventor in which gp 120 was synthesized from cells which had been infected with a recombinant vaccinia virus by means of which the env is expressed, showed that gp 120 is not formed in a stable manner in oligomeric form. The test results indicate that gp41 must be present during the biosynthesis of the env glycoprotein in order to assure the oligomerization. The domain of gp41 responsible for this oligomerization is probably located within the 129 amino terminal amino acids of gp41 (Earl, P. L. et al. "Proc. Natl. Acad. Sci. USA", 87, 648-652 (1990)).
Since gp41 is obviously of great significance in the synthesis of native gp160, experiments have also been undertaken to produce the complete gp160 in stable form. Barret et al. ("AIDS Research and Human Retroviruses" 5, (2), 159-170 (1989)) obtain gp160 e.g. by expressing the env gene in eukaryotic cells and subsequent purification; however, in addition to detergents they also use potassium thiocyanate, which has a strong chaotropic action and denatures proteins. Papers of the author have shown that the pure preparation of gp160 posed great problems for the following reasons:
Gp160 is, as described above, a membrane protein. Detergents are necessary for dissolving this protein out of the membrane. They are also necessary since isolated gp160 molecules aggregate in the absence of detergents. However, detergents result in a change of the molecule. It was determined that gp160 with detergents exhibits a change of the protease digestive pattern in comparison to the untreated protein. When the detergent is removed out of the solution, which is necessary at the latest when the protein is to be injected into a patient e.g. for the purpose of vaccination, the oligomer decomposes to the monomer. For the reasons cited, the chances of being able to produce pure gp160 in native form appear to be very slim.
The essential disadvantage which all previous methods for developing a vaccine against HIV on the basis of the isolated ectodomain of the env glycoprotein have in common is thus most likely the fact that the protein used as immunogen was not present in native and oligomeric form. This becomes clear from the fact that the titers indicated by the authors, which were determined by ELISA, are very high (around 1/100,000) and on the other hand the titers measured in the neutralization test are extremely low (often far less than 1/1000). This suggests that the antigens used in each instance do exhibit a strong immunogenic action, thus, a large amount of antibodies are formed against them, but that these antibodies hardly recognize or do not recognize at all the native protein and therewith the virus either.
The low titers in the neutralization test show that antibodies recognizing the native antigen are present only in very low titers. However, low-titer antibodies frequently result in an elevation of the virus infectivity (Prabhakar et al., Nature, 290, 590 (1981)). In other words, instead of protecting the host from the infection, the infectivity is increased by virus-specific immunoglobulins. In this instance the vaccine has the opposite of the desired effect as a consequence. The problem described also results in the case of all other viruses comprising a surface membrane with membrane-bound proteins.
In addition to their use as vaccine native proteins and protein domains are also used as immunotherapeutic agent or for diagnostic purposes. For diagnosis both antigens and also the antibodies directed against them are used.